Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca2+ and cGMP. Light stimulates the hydrolysis of cGMP and the resulting fall in [cGMP] leads to closure of cGMP-gated channels and a drop in Ca2+. The two key enzyme systems involved in regulation of cGMP and Ca2+are a cGMP-specific phosphodiesterase (PDE) and guanylate cyclase (GC). PDE is a multisubunit enzyme (PDEalphabetagamma2delta2) and peripherally membrane associated, while GC is a single subunit integral membrane protein. The Ca2+ sensitivity of photoreceptor GC is mediated by Ca2+-binding proteins termed GCAPs (guanylate cyclase-activating proteins). The regulation of GCs by GCAPs, the complexity of which is enhanced by the presence of at least three GCAPs and two GCs in human retina, is incompletely understood. This application aims to investigate outstanding and unsolved questions in the regulatory processes regarding PDE and GC/GCAPs. Specific Aim 1 addresses unsolved problems in PDE research, particularly the identification of chaperones essential for the correct folding of nascent polypeptides during biosynthesis, and elucidation of the role of Pdelta, a putative fourth subunit, by in vitro and in-vivo expression. In Aim 2, we propose to address mechanisms leading to dominant cone dystrophy when mutant GCAPs are expressed in the retina of transgenic mice. Additional questions concern the roles of GCAP2 and GC2 in phototransduction. GCAP1,2 double knockouts revealed a phenotype of delay in dark adaptation consistent with loss of activators. We showed (see preliminary results/progress report) that transgenic GCAP1 rescues the GCAP1/2 double knockout phenotype, suggesting that GCAP1 alone may regulate both rod and cone phototransduction. Elucidation of specific roles, particularly for GCAP2 and GC2, in the retina is part of Specific Aim 3. Taken together, this research will enhance our knowledge of the structure and function of components involved in the regulation of cGMP and Ca 2+, two important secondary messengers of phototransduction.